Thank You

There are other topics to write about, as promised at the beginning of this series.
Being that keeping a blog takes time and soon becomes a boring activity, I have decided to give you links instead of new pages. All the articles in the list below have been written by myself over the last couple of years. I could reorganize them into a more organic and readable form, but the concepts are already clear enough.

• Manual Corrections
• Automatic Baseline Correction
• Automatic Integration
• Perfect-Looking Integrals
• Visual Weighting
• 2D Phase Correction
• Further Hints for 2D Phase Correction
• 1D Phase Correction
• Automatic Phase Correction
• Non Linear Methods

I'll continue the blog when I find something new to say. I thank you all that have been reading me daily during the last month. Yesterday the blog received 84 visits, which was the July record.

Torrents of Cracked NMR-Warez

My most popular post, in two years of activity, has been the infamous "TopSpin NMR Free Download". That single (short) post has received as many comments as the rest of blog (sob!). People were so prompt to fix their anger in words but nobody was touched by the idea of starting a discussion. Unless you call discussion an exchange of flames. I have learned the lesson, in my own way. This time I have chosen a more explicit title, so nobody can say he arrived here with pure and saint intentions.
I myself haven't changed my mind. Year after year, a lot of money is spent to acquire/upgrade NMR instrumentation and software. Attending a conference is not cheap and renting a booth there is really expensive. The number of computers sold keeps increasing year after year. How is it possible that there is so much money in circulation and, at the same time, people cry because they can't pay an NMR program (but could nonetheless find the money to buy a new computer...) ?
Our society spends a lot of money to buy goods that are never used, or misused, or are notoriously dangerous for the health or the environment. If you really want to buy a thing that you aren't going to use, don't buy a computer. Just think at the environmental cost of disposing of it in a few years to come. Don't buy a cell phone. Your drawer is already full of old items you don't know what to do with. Don't buy a book. Just think at how many unread books are accumulating dust in your library. Buy software in digital form. It's a wiser form to waste your money.


If you came to this blog by accident, would you please have a look here around and try reading any other post, just to develop a more correct idea of what this blog is about?
In the last 6 weeks I have been publishing an article per day, with few exceptions. My activity has had no effect on the traffic. For example, the blog was more visited in April, a month in which I wrote a single article in total.
Tomorrow I will therefore conclude the current series of daily articles.

Recipe to Remove the t₁-noise

Take a column of the processed 2D plot (column = indirect dimension). Use a mapping algorithm to identify the transparent regions (not containing peaks). In the following I'll call them the "noisy regions".
The idea is to delete these region. Setting all their points to zero would be too drastic and unrealistic. What you do, instead, is to calculate the average value in this region. More exactly, the average of the absolute values. At the end you have a positive number which is a measure of the noise along that column. Repeat for all the columns. At the end you have the values of noise for each column. Noise is higher when there is a big peak (on the diagonal or elsewhere). Noise is low where there's no signal. Annotate the minimum value for this noise, let's call it "min". Now, pick again each column. Divide its "noisy regions" by the value of their own noise, then multiply them by "min". The portions containing true peaks are not affected.
The result is that now all the columns show as much noise as the least noisy column. In other words, the noise is = min everywhere.
The merit of this technique is that the final spectrum looks extremely natural, even if it's not. You can't cancel a peak by accident, because nothing has been zeroed.
De-noising, as describe here, can be successfully combined with baseplane correction and symmetrization. Remember that baseplane correction comes first and symmetrization always come last.

Second Love

Since the beginning of this series of "lessons", the stress has been on 1-D processing. I have shown that 1-D processing can be as tricky and important as 2-D processing. If you are working exclusively with large bio-molecules you may well live with the sensation that there's no NMR with less than 2 dimensions. The NMR field is divided in many rooms and there's not enough communication among them. I have attended several NMR conferences where there was indeed the possibility for the groups, working in distant field, to merge their knowledge for a week. My impression is that every one keeps speaking his own language and keeps doing the same things for decades. NMR is not a unifying technique. The main link between the researcher is not their society, but the factory that builds the instruments.
Today I want to write about a couple of processing operations that are specific to 2D. Symmetry is the first example that comes to my mind. In homo-nuclear correlation spectroscopy, we expect that the two halves of the map, divided by the main diagonal, are the mirror image of each other. They aren't for a couple of reasons:

• The line-widths along the indirect dimension are generally larger.
• The t₁ noise is specific to t₁, as the name says.

The software can force the two halves to be the same. Every couple of corresponding points is compared. The point with the lowest absolute value is the "correct" point; the point with the highest absolute value is replaced. In the case of J-resolved spectra, the axis of symmetry is different, but the principle the same.
After the substitution, the resulting shape of the peaks is that of a pilaster, instead of a column. I prefer the original shape, but in many cases the advantages are overwhelming. My rule is easily stated: I symmetrize all my COSY. Both the COSY experiment and symmetrization looks terribly old and out of fashion, but they are so simple and fast (I mean: the gradient enhanced variant), that I always acquire a COSY, if I must acquire another 2D of the same sample. The COSY comes almost for free. They say that symmetrization might create false cross-peaks. This is right, yet it's a rare event and not a dangerous one. If you have some experience you can usually recognize the false cross-peaks. If you haven't, you can compare the symmetrized and unsymmetrized versions of your COSY. It takes a few seconds.
I don't symmetrize my phase-sensitive spectra, unless the noise is exceptionally intense. In the phase-sensitive case I feel that the original shape of the peaks carries precious information (like a fingerprint), and that a baseplane correction is enough to clean the spectrum. You can combine baseplane correction and symmetrization, only in this order. Another trick, which Carlos taught me, can remove the "t₁ noise". Actually it's a modification of the baseplane correction and it's more an aesthetic trick than an operation to clean the spectrum. Tomorrow I'll describe my personal implementation of it.

Report Generator

The natural complement to the Multiplet Analyzer is a Report Generator. The former starts from the list of frequencies (the output of peak-picking) and generates a table of chemical shifts and couplings. The Report Generator starts from this table and generates a formatted list, ready to be inserted into a patent or an article; the format is compliant with the rules dictated by the patent office or by the receiving journal, etc..
It's counter-productive, for a software vendor, to explain all this details and intermediate stages. It's more impressive to state that a program can start from the FID and automatically generate the article (and maybe even sending it via email to the editor of the JOC!). If such a monolithic thing really exists, it would be a case of bad design, but the marketing appeal can't be argued.
Selling a program is easy. Convincing the customer to use it, that's difficult!

We have seen that the multiplet analyzer is limited to first-order signals. We have also examined other wonderful weapons at our disposal: the simulation of spin systems, to extract the NMR parameters from second-order multiplets, and deconvolution, to untangle overlapped signals.
A software vendor can't say: "Our research team is working hard to deploy a New Integrated Software Solution (TM) that automatically solves the most difficult cases" and in the meanwhile leave the user alone. The customer needs to publish his article right now, he can't wait for the next release of the software.
We have 3 established, long-standing and effective methods. They can be applied to different regions of the same spectrum. Let the Report Generator act as a central server, capable of accepting input by any method, even those that I am forgetting now, and of sending out the results in many different formats.

Suggested Reading:
The J Manager as a Center of Gravity.
This tutorial can be followed in practice, because it includes the same sample files that are shown into the pictures. There are also step-by-step instructions. The concept should be clear.

Multiplet Analyzer

Not all the spectra are second order. Actually, today's marketing insists to generalize that, in our new century, almost all the spectra are first order. Such a generalization makes sense if you process a spectrum (or less) per year, otherwise your destiny is to meet, sooner than you expect, something second-order. It's true that you are allowed to describe your spectrum as a sequence of generic multiplets, avoiding a more detailed analysis. This is tolerated but hasn't become the recommended practice yet. That said, there are certainly a lot of first-order multiplets in our spectra and extracting shifts and J it's an easy but tedious task. Can the computer help us? See for example the following spectrum of 1-pentyne in CDCl₃.

(The peak at 1.56δ is an impurity). There are, from left to right, a triplet of doublets, a triplet, a sextet and another triplet. The manual extraction of parameters is easy. Let's start from the sextet, because it's a curious rarity. It's enough to know the frequencies of the two external lines. The distance, divided by 5, gives the J. The sum, halved, gives the chemical shift. If you use a pocket calculator, there is the risk of a wrong transcription of the values from the monitor to the calculator and from the calculator back to the computer (for example into MS Word). The risk is low, but not zero. Besides this risk, the whole operation is time-consuming. Finally, it's not cool: you have to move cyclically from the NMR software to the pocket calculator to MS Word, etc...
Being it a simple operation, it can be performed automatically by the computer. It will not start directly from the data points, but from the same values used by an operator: the list of frequencies (peak-picking), the intensities at these frequencies, the list of integrals. These lists can also be generated automatically; in conclusion the whole process can be performed in automatic fashion. In practice, however, it's not convenient. Why? Suppose that the maker of the program claims that the automatic method works in the 99.9% of the cases. That's a generous claim, difficult to trust. For example, if the program doesn't recognize our impurity at 1.56δ as such (see picture above), it will fail to recognize the multiplet as a sextet. But, even if we believe the claim, how do we know if our spectrum belongs to the 99.9% or to the 0.1% of failures? We are forced to check the output with care. The time saved with the automatic processing will be lost for the check.
In my opinion, it's best to perform the integration and the peak-picking first (manually or automatically) and perform the check at this stage: the inspection is visual, not textual, therefore faster. In the case shown by the picture, this is the moment to remove the entry "1.56" from the list of frequencies. When all is ready, proceed with the computer-assisted extraction of the NMR parameters. A picture is more explanatory. Refer to your software or give a glimpse to this tutorial.
Although not a rule, Multiplet Analyzers ignore and remove the roof effect. Take for example our sextet. It can be recognized as such only if the intensity ratio is 1:5:10:10:5:1. The leftmost multiplet also has 6 lines, but the intensity ratio is 1:1:2:2:1:1, therefore it's recognized as a triplet of doublets. The roof effect prevents the recognition, therefore it's removed by averaging (symmetrization). For example, if the ratio of a triplet is 0.9:2:1.1, the computer calculates the average of the outer lines and the result is the theoretical 1:2:1. There's a notable exception. When there are two lines only, a single solution is possible, the doublet. When the lines are three, if we assume that all nuclei have spin = 1/2, the solution is still unique.
The roof effect can also exploited, in these cases at least. Like the old textbooks say, the chemical shift of a doublet doesn't correspond to the middle frequency, but to the center of mass. This is the single case I know when a multiplet analyzer performs a second-order analysis. I don't know if the center-of-mass rule is general and if all today's program observe it.
There's another, more obvious, rule to follow: any two coupling partners must show an identical splitting. In practice, the values extracted from two multiplets are rarely identical. Only the user can decide what to do. She can either:

1. Substitute the original values with an average value.
2. Remove what appears to be the less accurate value and put in its place the splitting shown by the partner.

Turning Point

The turning point in dynamic NMR was the article "DNMR: the program" by Gerhard Binsch (JACS, March 12, 1969). The article said: if we monitor chemical exchange by the coalescence of two singlets, a large variation of the rate is reflected into a small change of the spectrum. Therefore our estimate will be inaccurate.



If, keeping A and B as they are, we simply add a third nucleus C, coupled with both A and B, see what happens:



Now a small variation in the rate of exchange is the cause of a large change in the spectrum. Therefore we can estimate the rate (by simulation) with higher accuracy and confidence.
40 years later the lesson has not been learned yet and there's people who prefer to add a methyl group to their compounds to monitor the exchange rate by the coalescence of the singlets. I can understand this choice if the reason is to maximize the intensity of the signals.
I suspect, however, that the true reason is a different one. There is an approximate formula from which you can calculate the rate of exchange from the temperature of coalescence.
It's very approximate, but much easier to put in practice. A single spectrum and a simple formula instead of collecting 10 spectra and fitting each element of the series, then plotting the data to extrapolate the Eyring equation... The accurate method not only is much faster, it also avoids using a computer. The problem, today, is never the computer but always the software. Is the 40 years old DNMR difficult to obtain? You don't need DNMR: today you have WinDNMR, Spinworks, Mexico, iNMR....